J. Agric. Food Chem. 1993, 41, 858-862
050
Study of Nicotine Demethylation in Nicotiana otophora Ralph L. Chelvarajan, Franklin F. Fannin, and Lowell P. Bush' Department of Agronomy, University of Kentucky, Lexington, Kentucky 40546-0091
Microsomes from Nicotiana otophora catalyze the demethylation of nicotine to nornicotine in the presence of NADPH and oxygen. Activity was maximal a t pH 7.0-7.5and 30 "C. The enzyme appeared to be most stable in the presence of both nicotine and NADPH. Phosphate, magnesium, nornicotine, and concentrations of microsomal protein higher than 2 mg/mL were inhibitory. Typical rates of nornicotine formation were 10-50 pmolmin-' (mg of protein)-', while V,, and the apparentKm(nicotine) were estimated to be 105 pmol/min and 51 pM, respectively. Early studies suggest that Triton X-100 may be able to solubilize active nicotine demethylase. Nicotine demethylation satisfies some of the primary criteria for cytochrome P-450 involvement, including inhibition by anticytochrome P-450 reductase, but inhibition by carbon monoxide was not demonstrated.
INTRODUCTION Nornicotine is a major alkaloid in Nicotiana and is the principal alkaloid in 30-40% of the species (Bush and Crowe, 1983). Virtually all nornicotine is formed from N-demethylation of nicotine (Dawson, 1951; Bush, 1981; Leete, 1984), and depending on the species, much of the nornicotine accumulation in Nicotiana occurs as the leaf matures (e.g., N. otophora) (Dawson, 1945) or during curing (e.g., N. tabacum) (Wada, 1956). The demethylation of nicotine has been described to be controlled by one of two dominant genes (Griffith et al., 1955). This demethylation is not specific for the naturally occurring stereoisomer of nicotine, and partial racemization takes place during the process (Kisaki and Tamaki, 1961). The demethylation of nicotine as the primary source of nornicotine was first reported over 45 years ago by Dawson (1945). Nevertheless, the enzyme catalyzing this process has not been characterized or isolated. Schroter (1966) did detect in vitro nicotine demethylation in extracts from N. alata, but he did not present any in vitro properties of the enzyme catalyzing this process. The objective of this study was to characterize the in vitro properties of nicotine demethylation from N . otophora. MATERIALS AND METHODS Plant Material. N. otophora readily converts nicotine to nornicotine (Saitoh et al., 1985) and was used as the enzyme source. Lamina or pith was obtained from 6-month-old plants grown in a greenhousewith supplementallight from high-pressure sodium lamps. Preparation of Microsomes. Microsomes were isolated at 4 "C by a modification of methods used for the extraction of microsomesfrom higher plants (Reichhart et al., 1980;Dohn and Krieger, 1984; Mougin et al., 1990). Pith or lamina tissue was disrupted separately in a Waring blender in the presence of 8 volumes of N-(2-hydroxyethyl)piperazine-N'-[2-ethanesulfonic acid] (HEPES) buffer (50 mM, pH 7.5) supplemented with 250 mM sucrose,polyvinylpolypyrrolidone (PVPP)(0.2 g/g of tissue), and 15 mM mercaptoethanol. The crude extract was filtered through two layers of cheesecloth to remove undisrupted tissue, and the filtrate was centrifuged for 20 min at lOOOOg to remove cellular debris. The supernatant was subjected to further centrifugationat loooOOg for 60 min to pellet the microsomal fraction. The microsomal pellet was resuspended with the aid of a tissue homogenizer in 3 volumes of HEPES buffer (50 mM)
* Author to whom correspondence should be addressed. 0021-8561/93l1441-0858%04.00/0
supplementedwith glycerol (30%v/v) and was stored in aliquota of 200 pL at -80 "C. Nicotine Demethylation Assay. Nicotine [pyrrolidine-2W] was obtainedfrom NEN Research Productsand had a specific activity of 42.4 mCi/mmol. Routine enzyme assay consisted of approximately80 pg of resuspended microsomalprotein,20 pmol of NADPH, and 10 pmol of nicotine and was made up to 40 pL with 50 mM HEPES buffer of pH 7.5. NADPH was omitted for control incubations. The reaction was carried out at 30 "C for 30 min and was stopped with addition of 4OpL of 95% methanol, which also contained 50 mM nornicotine and nicotine to increase the recovery of [14C]nornicotineand [14C]nicotine.The amount of demethylation was measured using the thin-layer chromatography procedure of Fannin and Bush (1992). CytochromeP-450ReductaseAssay. The cytochromeP-450 reductaseactivitywaa determined by observing the reduction of cytochromec with NADPH at 550 nm (Dohnand Krieger, 1984). Oxygen Exclusion. Two treatments were carried out to determine if nicotine demethylation had a requirement for oxygen. The reaction components were dispensed into six 2.5mL vials which were then hermetically sealed. Four vials were flushed with nitrogen (60 mL/vial), and two of these were then flushed with air (60 mL/vial). The two control vials were not flushed with either gas. Solubilization. A number of detergents were screened for their ability to solubilize active nicotine demethylase from the microsomal fraction. About 100 p L of microsomal extract was incubated with 33.3 pL of various concentrationsof detergent. The control had no detergent. The detergent-protein mixture was incubated at room temperature for 20 min and then centrifuged for 60 min at 1OoooOg. An aliquot of the supernatant was carefully removed and assayed for nicotine demethylase activity. The supernatant was then discarded, and the pellet was resuspendedin buffer with the aid of an automaticdispensing pipet and then assayed for enzyme activity. RESULTS AND DISCUSSION Powdering excised leaf tissue in liquid nitrogen and then extracting the soluble proteins with buffer (Frear et al., 1969)resulted in yields of active microsomal preparations that varied greatly between preparations. Omitting the liquid nitrogen step and disrupting tissue in 8 volumes of extraction buffer increased the yield and reduced the variability. Browning of the crude extract could not be prevented by the addition of 0.2 gig PVPP or by a similar concentration of Amberlite XAD-4. As 15 mM mercaptoethanol did prevent browning, the extraction was performed in the presence of mercaptoethanol. A small amount of demethylation was detected in the supernatant from the lOOOOOg centrifugation. The su0 1993 American Chemical Society
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Nlcotlne Demethylation 5.1
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Figure 1. Relationship between microsomal protein concentration and demethylation. Protein concentrationwas measured using Coomassie protein assay reagent.
Figure 2. pH profile of nicotine demethylase. pH was varied using MES (pH 5.0-6.5) and HEPES (pH 7.0-8.5) buffers. Data are shown with a spline curve fit.
pernatant activity was not diminished by incubating the microsomal preparation in the absence of NADPH, at 4 or 55 "C, or by boiling the extract for 2 min prior to assaying. Hence, it was concluded that the demethylation associated with the supernatant was not enzymatic and was not characterized further. Although Schr6ter (1966)did not present any data on the in vitro characteristics of the demethylation of nicotine, the activity he measured may have been the nonenzymatic activity we observed in the lOOOOOg supernatant. Schriiter's activity too was located in the supernatant. Microsomes stored for up to 5 months at -80 "C were still able to catalyze nicotine demethylation. Unlike the enzymes catalyzing the N-demethylation of substituted 3-(phenyl)-l-methylureasin cotton (Frear et al., 1969), nicotine demethylase was not inactivated after one freezethaw cycle, but instead increased by up to 80%. The level of microsomal N-demethylase in N. otophora [le50pmol min-I (mg of protein)-'], as measured by ita capacity to demethylate nicotine, was considerablyhigher than N-demethylases from 12 species of higher plants. The levels of these N-demethylases [0.15 mM) and hydrogen peroxide (>4 mM) actually inhibited demethylation. Flushing vials with nitrogen completely inhibited the demethylation of nicotine, but if the nitrogen were immediately removed by flushing with air, activities comparable to the control values were obtained. These results indicate that the inhibition by nitrogen was purely due to oxygen depletion. Tetcyclasis, an inhibitor of cytochrome P-450 type enzymes (Canivencet al., 1989),a t 60 pM inhibited nicotine demethylation by 50 % . However, in wheat microsomes a concentration of only 10 pM resulted in an inhibition of over 80 % of cytochromeP-450 dependent aryl hydroxylase activity (McFadden et al., 1989). Carbon monoxide also inhibits P-450 enzymes, and this inhibition can be reversed by light of 450 nm (Donaldson and Luster, 1991). Sparging the reaction componentswith carbon monoxide for 60 s (Omura and Sato, 1967) caused a reduction in nicotine demethylation activity of only 20 % in lamina microsomes and no reduction in pith microsomes (Figure 6). White light (2000 pEinstein m-2 s-l) from a tungsten-halogen lamp did not reverse the slight inhibition observed in lamina microsomes, but further inhibited activity,possibly by interacting with the pigments present, resulting in the production of inhibitors to nicotine demethylation. Various concentrations of a polyclonal antiserum raised in a rabbit against H. tuberosus NADPH-cytochrome P-450 reductase were added to the routine nicotine demethylase assay. The control was the incubation in the presence of a corresponding concentration of nonimmunized serum. Increased concentrations of antiserum increased inhibition of nicotine demethylation (Figure 7), suggesting a role for cytochrome P-450 reductase in the demethylation reaction. On the basis of their inhibition studieswith the antiserum to cytochrome P-450 reductase, Benveniste et al. (1989) have concluded that cytochrome P-450 reductase is involved in reactions catalyzed by cytochromes P-450 in higher plants. Thus, if cytochrome P-450 reductase is involved in nicotine demethylation,this is a strong argument for the involvement of cytochrome P-450 as well. Cytochrome P-450 dependent monooxygenases have previously been reported to be involved in higher plant alkaloid metabolism (Madyastha et al., 1976) and may
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Figure 7. Effect of an antibody raised against H.tuberosus
cytochrome P-450 reductase on the demethylation of nicotine. also be involved in the demethylation of nicotine in N . otophoru. Nicotine demethylation,like cytochrome P-450 mediated reactions, occurs in the microsomal fraction and is dependent upon molecular oxygen and reducing equivalents provided preferentially by NADPH and to a lesser extent by NADH. The stimulation of nicotine demethylation a t subsaturating concentrations of NADPH by NADH is a characteristicof a number of cytochromesP-450 (West, 1980). Although only partial inhibition by tetcyclasis and carbon monoxide suggests that cytochrome P-450 may not be involved in nicotine demethylation, these results do not rule out cytochrome P-450 involvement. Previous studies have indicated that the association between carbon monoxide and different cytochromesP-450 varies and can depend on the presence or absence of substrates (Tuckey and Kamin, 1983; Schroder and Diehl, 1987). It is also possible that tetcyclasis could have been bound to microsomal lipid, lowering the free concentration of tetcyclasis. A t least one cytochrome P-450 dependent N demethylase in wheat (Mougin et ul., 1991) is known to be less sensitive to tetcyclasis than nicotine demethylase. The ability of peroxides to support demethylation in the absence of NADPH is not cited as a higher plant cytochrome P-450 characteristic by West (1980) but was considered in our study as certain cytochromes P-450 have been shown to be able to utilize peroxy compounds instead of NADPH (Estabrooke et al., 1984; Hollenberg et al., 1985). More cytochrome P-450 tests have to be carried out on the demethylation of nicotine before a definitive statement can be made on the role of cytochrome P-450, as not all of the primary criteria are satisfied by any one system (West, 1980). Senescent leaves are characterized by a degradation of chlorophyll, carotenoids, starch, and chloroplast proteins (Long and Weybrew, 1981). Yet, the level of nornicotine in the leaves of numerous Nicotiana species rises during the last stages of curing (Wada, 1956). Other N-demethylases have also been reported to become more active during senescence (Fonne-Pfister et al., 1988). In vivo results from our laboratory have demonstrated greater levels of nicotine demethylation activity in leaves of N . syluestris that were treated with ethylene to hasten senescence (Fannin and Bush, 1992). This suggests that the enzyme responsible for nornicotine synthesis is not degraded during early senescence but that the enzyme activity may be induced or activated during senescence. ACKNOWLEDGMENT We thank Dr. I. Benveniste and Dr. F. Durst of CNRS, Strasbourg, France, for kindly supplying the antiserum to the H. tuberosus NADPH-cytochrome P-450 reductase.
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